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The crystal structure studies of a second conformational state of Clostridium symbiosum pyruvate phosphate dikinase (PPDK) are reported. The studies confirm the swivel motion of the His domain adjacent to the phosphoenolpyruvate (PEP)-binding domain.

Human hyaluronidase-1 is implicated in cancer proliferation, angiogenesis and inflammatory diseases. Its expression is up regulated in advanced stages of bladder cancer, whereas the expression of the alternative splice variants is down regulated.

Contributions made by Pseudomonas aeruginosa (PaADI) core residues Cys406, His278, and Asp166 and the contribution from Asp280 to catalysis of the formation and hydrolysis of the Cys406-alkyluronium intermediate were assessed by kinetic analysis of site-directed mutants. The results suggest that electrostatic interactions play a dominant role in PaADI catalysis.

Study was conducted to define the chemical function, a new member of the isocitrate lyase enzyme family derived from the flowering plant Dianthus caryophyllus. A substrate screen that employed a (2R)-malate core structure was designed on the basis of an analysis of the structural contexts of sequence markers common to the C-C bond lyases of the isocitrate lyase/phosphoenolpyruvate mutase superfamily.

Expression of PSR132 protein from Dianthus caryophyllus was induced in response to ethylene production that was associated with petal senescence and therefore the protein is named petal death protein (PDP). The crystal structure of PDP was determined at 2.7 angstrom resolution, indicating that a dimer-of-dimers oligomeric association.

Research has been conducted on Mytilus edulis PEP mutase. The crystal structure of this mutase in complex with the substrate-analogue inhibitor has been investigated and the results indicate that the phosphoryl group transfer occurs on PEP Si-face with the configuration retention at phosphorus.

Structural analysis of the enzyme-substrate complex reveals that phosphonopyruvate binds in the alpha/beta-barrel's central channel of the Beta-strands C-termini. The structure resembles that of pyruvate kinase but differes in the acid/base catalysis function.

Research on a mutant form of beta lactamase from Staphylococcus aureus shows that substrate specificity is not determined at the acyl-enzyme state. This was the conclusion of researchers who studied the binding modes of benzylpenicillin and cephaloridine.

The mechanism of phosphoenolpyruvate mutase (PEP mutase) catalysis is discussed relative to site-directed mutagenesis studies of active site residues. The sequencing, expression, cloning, and mutagenesis of the Mytilus edulis PEP mutase gene in Escherichia coli are covered. A catalytic motif has been proposed. It could serve for both phosphoryl transfer and the C--C cleavage enzymes of the PEP mutase superfamily.

Research was conducted to identify the specificity requirements for phosphorylation of Mycoplasma capricolum HPr. A mutagenesis method was used in which residues 43-57 in a chimera were replaced by the Escherichia coli sequence. Results indicate that the kinase-HPr interaction takes place mostly through a hydrophobic region. Comparisons conducted between the chimeric construct and M. capricolum provide a basis for the interpretation of activity assays results.

The effect of the omega-loop deletion in the function and structure of the enzyme beta-lactamases was investigated. A beta-lactamase mutant protein was produced using the four-primer overlap-extension method. A double-stranded plasmid DNA was then used to resequence the beta-lactamase gene. It was found that the absence of the omega-loop eliminated Glu166, a primary catalytic residue, thus resulting in dysfunctional deacylation. It also eliminated the enzyme's ability to hydrolyze beta-lactam compounds.

The presence of a non-proline cis peptide between Glu166 and Ile 167 in the active site of beta-lactamase from Staphylococcus aureaus PC1 was determined. A site-directed mutant N136A was produced to examine the role of the interaction between the side chain of Asn136 and the main chain of Glu166. No measurable hydrolytic activity towards penicillins or cephalosporis by the enzyme was observed, apart from the chromegenic cephalosporin, nitrocefin. The hydrolysis of nitrocefin after reincubation with several beta-lactam compounds was monitored, and cephalosporins were found to form stable acyl complexes with the enzyme.Electrospray mass spectrometry was used to determine the molecular weight of the mutant.

The structure and enzymatic functions of beta-lactamase from Staphylococcus aureus PC1 was evaluated through construction of two circularly permuted proteins, cp228 and cp254. Both have an eight residue long peptide that joins the N- and C- terminal helices of the native molecule with N-terminal residue located on positions 228 and 254, respectively. Cp228 has shown a decrease of activity towards beta-lactam antibiotics and a greater activity towards cefataxime.

Two site-directed mutant enzymes of the class A beta-lactamase from Staphylococcus aureus PC1 were produced to block the site occupied by a hydrolytic water molecule in the native enzyme. The crystal structures of these two mutant enzymes, N170Q and N170M, were determined. Results indicated that the water site is preserved and that Met170 occupies part of the space that is needed for substrate binding. The short contacts between the substrate and the enzyme may cause structure perturbation and inactivation.

Two mutant beta-lactamases from Staphylococcus aureus PC1 which probe key catalytic residues were formed by site-directed mutagenesis. The nucleophilic group that attacks the beta-lactam carbonyl carbon atom was removed in the S70A enzyme. The crystal structure of S70A beta-lactamase was determined at 2.1 angstrom resolution. The structure is similar to that of the native enzyme except for the mutation site. The K73H beta-lactamase was tested for the possible role of Lys73 in proton transfer. It did not show any activity toward benzylpenicillin. The crystal structure of K73H beta-lactamase was determined at 1.9 angstrom resolution.

Photoaffinity labeling, site-directed mutagenesis and structural analysis of pyruvate phosphate dikinase from Clostridium symbiosum reveals that the nucleotide binding site is present in the N-terminal. X-ray crystal structure studies indicates that the enzyme folds into N-terminal, central and C-terminal regions. ATP binds in a crevice formed by subdomains one and three in a manner similar to ADP binding in D-alanine-D-alanine-ligase.

A mutated form of troponin c from chicken skeletal muscle was used as a spectral probe for monitoring the Ca2+-induced structrural transition of the regulatory N domain. Phe-29 at the COOH-terminal fragment of the A helix near the Ca2+-binding loop of site I was replaced by tryptophan (trp) which elicited unambiguous spectral features owing to the absence of other trp or tyrosine. Alteration in spectral features was restricted to Ca2+-binding in the N domain conformational transition. The spectral feature of the probe mutant was also applied to the analysis of Ca2+-binding affinities of five double mutants wherein nonpolar amino acids were replaced by polar residues.

The crystal structure determination of recombinant GK[alpha][beta] and GK[beta][beta] from the marine worm Namalycastis sp. with a transition state analogue, [Mg.sup.2+]-ADP-N[O.sup.3-]-glycocyamine is described. The active site environments of the glycocyamine kinase (GK), homodimeric creatine kinase (CK), and monomeric arginine kinase (AK) at the bound states provide insight into the structural determinants of substrate specificity and the coupling between the structural asymmetry and negative cooperativity proposed for CK which is not supported in the case of GK.

Several studies are conducted to explain the substrate binding and stereoselectivity phenomena observed in the Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA), a member of the II zinc-dependent aldolase family. The polarization of the substrate for protein transfer in the FBPA compound is shown to be highly dependent on the zinc mobility within it.

The biochemical characterization and cystallographic analysis of HI0827 protein isolated from Haemophilus influenza Rd KW20 was carried out to examine the structural variations within the acyl-CoA thioesterase family. The findings provided insight into a new structural class of hexameric hot dog fold enzyme, acyl-coenzyme A (acyl-CoA) thioesterases.

A study was conducted to examine the biochemical and biological function of the hot dog fold thioesterase YciA (EcYciA) from Escherichia coli and its close sequence homologue HI0827 from Haemophilus influenzae (HiYciA). The results provide insight into significant role of YciA for efficient CoA-regulated hydrolysis of cellular acyl-CoA thioesters in a wide range of bacteria.

The isolation, structure and chemical mechanism for catalysis of the Pseudomonas aeruginosa, arginine deiminase (ADI) (L-arginine) which involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate is described. The findings from the studies would help to design the arginine analogs such as L-canavanine that migaht react with the ADIs to form inactive covalent adduct during catalytic turnover.

The crystal structure of PA4872 from Pseudomonas aeruginosa in complex with oxalate and [Mg.sup.2+] is studied. The results have shown that PA4872 is an oxaloacetate decarboxylase, which is used primarily by pseudomonas, and that the enzyme is used by differential bacterial species to maintain pyruvate and bicarbonate cellular levels in many pathways.

The structure and properties of recombinant phosphonopyruvate (P-pyr) hydrolase (PPH) from variovorax sp. Pal2 expressed in E. coli is reported. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop.

The two crystal structures of the C123S mutant of 2-methylisocitrate lyase have been determined, one with the bound reaction products. The structure-based sequence analysis and phylogenic tree construction reveal determinants of substrate specificity, highlight nodes of divergence of families, and predict enzyme families with new functions.

The results from transient-state kinetic experiments which show that arginine deiminase catalysis proceeds through a covalent adduct formed by activesite Cys406 displacement of ammonia from the guanidium carbon of L-arginine are presented.

The crystal structures of wild-type and D58A mutases, in the apo state and in complex with Mg(II), are presented. The members of the phosphoenolpyruvate (PEP) mutase/isocitratelyase (ICL) family share sequence homology and overall fold, and use similar modes of flexibility.

Research was conducted to examine the location of the phosphate binding site within Clostridium symbiosum pyruvate phosphate dikinase (PPDK). Arg337 was implicated as a key residue for P(sub i) binding by computer modeling studies and was tested by site-directed mutagenesis. Results indicate that the P(sub i) binding site is located within the crevice of the PPDK N-terminal domain, at a place that is flanked by both the Mg2+ cofactor and the ATP beta-P.